A recent external quality assessment (EQA) in Europe revealed that 60% of laboratories need to improve molecular Zika virus detection (6). Laboratories in affluent countries conduct Zika virus diagnostic testing predominantly in travelers returning from tropical regions. In resource-limited settings to which multiple co-circulating arboviruses are endemic, the diagnostic demands differ entirely. To evaluate the diagnostic landscape in the region most affected by Zika virus, we performed an EQA of molecular Zika virus diagnostic testing in Brazil during 2017.

The Study

Fifteen laboratories from 7 Brazilian states participated in this study; these laboratories are spread across ≈2,500 km longitude, including the areas most affected during Brazil’s Zika virus outbreak (1). Participants were university laboratories, hospital laboratories, federal research institutes supporting public health services, and a diagnostic testing company. We provided EQA panels to all laboratories. Each panel comprised 12 lyophilized samples containing inactivated full virus spiked into human plasma tested negative for arboviruses beforehand. The panel consisted of 4 Zika virus–positive specimens of 103–106 RNA copies/mL to assess sensitivity and determine viral load. Zika virus–negative specimens to assess specificity comprised dengue virus serotypes 2 and 4, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, and chikungunya virus at ≈105 50% tissue culture infective dose/mL each and a negative plasma specimen (Table 1). Moreover, each panel included the international World Health Organization (WHO) Zika virus standard for quantification (7). However, the WHO standard has limited availability. International shipment of this standard is subject to strong biosafety restrictions because it derives from live Zika virus. Therefore, we designed and acquired a Zika virus armored RNA (Asuragen, Austin, TX, USA). The Zika virus armored RNA is a synthetic RNA covering the target sites of 9 Zika virus–specific real-time RT-PCRs as described previously (3), encapsulated into bacteriophage proteins. This highly stable, noninfectious, pseudoviral particle can be used as a universal control for the covered assays, shipped without biosafety concerns, and used as a control for both nucleic acid preparation and RT-PCR.

We asked all laboratories to conduct molecular Zika virus diagnostics as routinely done with clinical samples and to quantify Zika virus–positive specimens using both standards. All but 1 laboratory used the same real-time RT-PCR protocol developed by Lanciotti et al. (2), highlighting the wide dissemination of this assay in Brazil and suggesting comparability of test results within this study (Table 1). We found no significant difference between samples containing comparable quantities of the Asian and the African Zika virus lineage, suggesting suitability of the protocols for both lineages (p = 0.313 by Fisher exact test).

Quantification of Zika virus loads did not differ significantly between use of the armored RNA and the WHO Zika virus standard, with only 0.76 log10median deviation between results (p = 0.429 by Wilcoxon signed rank test). This observation suggests usability of the armored RNA for Zika virus quantification in tropical regions. Irrespective of the standard, viral load determinations among laboratories were comparable with 0.12–0.88 log10median deviations of viral load estimates among laboratories for individual Zika virus specimens. However, we also observed drastic deviations of up to 6 orders of magnitude (Figure 2), suggesting that caution must be taken upon comparing viral load determinations as markers for severe Zika virus disease (8,9) among different laboratories.

Conclusions

Some laboratories in Brazil showed suboptimal sensitivity and specificity of Zika virus diagnostic testing. However, these laboratories performed comparably to those in Europe (6). Neither sensitivity nor specificity differed significantly between laboratories in Brazil compared with those in Europe (p = 0.767 and p = 0.324, respectively, by Fisher exact test). Similarly, the proportion of perfectly performing laboratories in this EQA (27%) was comparable with previous EQAs of flavivirus molecular diagnostics, including yellow fever virus (18%), dengue virus (24%), and West Nile virus (27%) (10–12). Flavivirus molecular diagnostics are thus generally challenging and benefit greatly from controls, such as those provided in this EQA. This study underscores the need to combine RT-PCR and serologic testing in Zika virus diagnostic testing, despite their inherent limitations (3).

Independently of the challenges of Zika virus molecular detection, because of taxation and distributor margins, RT-PCR reagents in Latin America are usually 100%–200% more expensive than in affluent countries (13). Limited resources and relatively higher costs potentially force laboratories in Brazil to seek inferior, more affordable solutions. Enhanced access of laboratories in tropical regions to state-of-the-art reagents is thus an unresolved key component of outbreak response. Further EQAs in Brazil should involve state laboratories that carry a large proportion of Zika virus testing within the public health care system. Unfortunately, the state laboratories we contacted for this EQA could not participate because of limited resources.

Mr. Fischer is a PhD student at the Institute of Virology at Charité-Universitätsmedizin Berlin, Germany, and the German Centre for Infection Research, Germany. His main research interests are diagnostics of emerging arboviruses.

This work was supported by the German Centre for Infection Research through a fast-track program for Zika virus outbreak response to J.F.D. and partially supported by the European Union’s Horizon 2020 research and innovation program (ZIKAlliance, grant agreement no. 734548 to X.d.L. and EVAg, grant agreement no. 653316 to C.D.).